Electrofusion is the common name for procedures that induce fusion of living cells using electricity. Cell-cell electrofusion (CCE) is the generic term used to describe electrofusion of living cells. CCE can refer to fusion of one cell type to a different cell type, or it can refer to fusing cells of the same type. In this application, reference to fusion of cells is intended to encompass both fusion of different cell types and also fusion of the same cell types. Moreover, it is intended to encompass the fusion of two or more cells to each other.
CCE processes generally involve three principal steps. First, fusion partners (i.e., two or more cells to be fused to each other) must be forced into contact with each other between two electrodes or some other means of inducing electrofusion. The cells must be in an electrically conductive medium. Second, one or more electrical pulses are applied to the cells that are in contact between the electrodes. Electrical pulses induce fusion and are administered by creating and maintaining a potential (voltage) difference across the electrodes. CCE is usually achieved using direct current (DC) pulses. The third and final CCE step occurs naturally; fused cells anneal into one cell due to their normal fluidity. CCE processes do not normally yield 100% fusion. Typically, a fraction of the contacted cells are induced to fuse while the remaining fraction does not fuse. Also, many of the extensively used methods involve steps which have a high rate of cell killing.
Most existing commercial CCE devices and applications known to the applicants use a process called dielectrophoresis to cause cell-cell contact. Dielectrophoresis is the application of alternating current (AC) to cause fusion partners to line up in chains between the electrodes. Thus, cell-cell contact is achieved at the points where adjacent cells in a chain are touching. Dielectrophoresis is incorporated into the first step of the three-step fusion process described above. After chains have formed, one or more DC pulses are delivered to induce fusion and the cells are allowed to anneal.
Jaroszeski et al., (Biophysical Journal, Vol. 67, Oct. 1994, 1574-1581) discloses apparatus and methods developed to enable mechanically facilitated cell-cell electrofusion to be performed. The apparatus and methods mechanically place cells in contact before fusion. A novel fusion chamber is disclosed composed of two functionally identical electrodes that are housed in a multi-layer structure. The layers function as a support for the electrodes. They also allow adjustment of the distance between opposing electrode faces. The electrodes were constructed to allow cells to be deposited, by vacuum, onto each face. The electrode faces were positioned at a predetermined distance from each other to mechanically force cell-cell contact between the deposited cells. Fusion was induced by delivering direct current pulses to the juxtaposed cells.
Jaroszeski et al. (Analytical Biochemistry, 216, 271-275 (1994)) discloses a cytometric method for detecting and quantitating hybrid cells that resulted from cell-cell electrofusion. Cells from two different lines and two vital fluorescent dyes were used in conjunction with a flow cytometer to demonstrate the method.
The German Patent Publication DE 3505147 A1 to Strellrecht et al. discloses an electrofusion process wherein cells are fixed on a first and second carrier. The two carriers are arranged so that the cells that are fixed to the respective carriers are opposite relative to each other. The cells are moved toward each other forming pairs of cells, one from each carrier. The pairs are each fused.
It would be advantageous to provide more efficient and effective means for inducing cell-to-cell contact and fusion than that described above. The present invention provides improved means for inducing such cell-cell contact and uses electric pulses applied from a different direction relative to deposited cells than prior art.